The transcription factor CmLEC1 positively regulates the seed-setting rate in hybridization breeding of chrysanthemum

Abstract

Distant hybridization is widely used to develop crop cultivars, whereas the hybridization process of embryo abortion often severely reduces the sought-after breeding effect. The LEAFY COTYLEDON1 (LEC1) gene has been extensively investigated as a central regulator of seed development, but it is far less studied in crop hybridization breeding. Here we investigated the function and regulation mechanism of CmLEC1 from Chrysanthemum morifolium during its seed development in chrysanthemum hybridization. CmLEC1 encodes a nucleic protein and is specifically expressed in embryos. CmLEC1’s overexpression significantly promoted the seed-setting rate of the cross, while the rate was significantly decreased in the amiR-CmLEC1 transgenic chrysanthemum. The RNA-Seq analysis of the developing hybrid embryos revealed that regulatory genes involved in seed development, namely, CmLEA (late embryogenesis abundant protein), CmOLE (oleosin), CmSSP (seed storage protein), and CmEM (embryonic protein), were upregulated in the OE (overexpressing) lines but downregulated in the amiR lines vs. wild-type lines. Future analysis demonstrated that CmLEC1 directly activated CmLEA expression and interacted with CmC3H, and this CmLEC1–CmC3H interaction could enhance the transactivation ability of CmLEC1 for the expression of CmLEA. Further, CmLEC1 was able to induce several other key genes related to embryo development. Taken together, our results show that CmLEC1 plays a positive role in the hybrid embryo development of chrysanthemum plants, which might involve activating CmLEA’s expression and interacting with CmC3H. This may be a new pathway in the LEC1 regulatory network to promote seed development, one perhaps leading to a novel strategy to not only overcome embryo abortion during crop breeding but also increase the seed yield.

References

[1] Kaul, M. L. H. Male Sterility in Higher Plants. Monographs on Theoretical and Applied Genetics (Springer, 1988).

[2] Zhou, W. et al. A matter of life and death: molecular, physiological, and environmental regulation of seed longevity. Plant Cell Environ. 43, 293-302 (2019).

[3] Amlou, M., Pla, E., Moreteau, B . & David, J. R. Genetic analysis by interspecific crosses of the tolerance of drosophila sechellia to major aliphatic acids of its host plant. Genet. Sel. Evol. 29, 511-522 (1997).

[4] Rojas-Barrera, I. C. et al. Contemporary evolution of maize landraces and their wild relatives influenced by gene flow with modern maize varieties. Proc. Natl Acad. Sci. USA 116, 21302-21311 (2019).

[5] Burgess, M. B. et al. Effects of apomixis and polyploidy on diversification and geographic distribution in Amelanchier (Rosaceae). Am. J. Bot. 101, 1375-1387 (2014).

[6] Tonosaki, K. et al. Overcoming the species hybridization barrier by ploidy manipulation in the genus Oryza. Plant J. 93, 534-544 (2018).

[7] Verdier, J. & Thompson, R. D. Transcriptional regulation of storage protein synthesis during dicotyledon seed filling. Plant Cell Physiol. 49, 1263-1271 (2008).

[8] Roscoe, T. T. et al. Complementation of seed maturation phenotypes by ectopic expression of ABSCISIC ACID INSENSITIVE3, FUSCA3 and LEAFY COTYLEDON2 in arabidopsis. Plant Cell Physiol. 56, 1215-1228 (2015).

[9] Prieto-Dapena, P. et al. Seed-specific expression patterns and regulation by ABI3 of an unusual late embryogenesis-abundant gene in sunflower. Plant Mol. Biol. 39, 615-627 (1999).

[10] Horstman, A. et al. The BABY BOOM transcription factor activates the LEC1-ABI3-FUS3-LEC2 network to induce somatic embryogenesis. Plant Physiol. 175, 848-857 (2017).

[11] Cernac, A. et al. WRI1 is required for seed germination and seedling establishment. Plant Physiol. 141, 745-757 (2006).

[12] Alejandro et al. Arabidopsis LEC1 and LEC2 orthologous genes are key regulators of somatic embryogenesis in Cassava. Front Plant Sci. 10, 673 (2019).

[13] Kumar, V., Jha, P. & Van Staden, J. LEAFY COTYLEDONs (LECs): master regulators in plant embryo development. Plant Cell Tissue Organ Cult. 140, 475-487 (2020).

[14] Jo, L. et al. Combinatorial interactions of the LEC1 transcription factor specify diverse developmental programs during soybean seed development. Proc. Natl Acad. Sci. USA 117, 1223-1232 (2020).

[15] West, M. et al. LEAFY COTYLEDON1 is an essential regulator of late embryogenesis and cotyledon identity in arabidopsis. Plant Cell 6, 1731-1745 (1994).

[16] Chen, W. et al. A CCAAT-binding factor, SlNFYA10, negatively regulates ascorbate accumulation by modulating the D-mannose/L-galactose pathway in tomato. Hortic. Res. 7, 200 (2020).

[17] Kagaya, Y. et al. LEAFY COTYLEDON1 controls seed storage protein genes through its regulation of FUSCA3 and ABSCISIC ACID INSENSITIVE3. Plant Cell Physiol. 46, 399-406 (2005).

[18] Baumbusch, L. O. et al. LEC1, FUS3, ABI3 and Em expression reveals no correlation with dormancy in Arabidopsis. J. Exp. Bot. 55, 77-87 (2003).

[19] Carbonero, P., Iglesias-Fernández, R. & Vicente-Carbajosa, J. The AFL subfamily of B3 transcription factors: evolution and function in angiosperm seeds. J. Exp. Bot. 1, 871-8808 (2017).

[20] Santos-Mendoza, M. et al. Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis. Plant J. 54, 608-620 (2008).

[21] Tan, H. et al. Enhanced seed oil production in canola by conditional expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in developing seeds. Plant Physiol. 156, 1577-1588 (2011).

[22] Lepiniec, L. et al. Molecular and epigenetic regulations and functions of the LAFL transcriptional regulators that control seed development. Plant Reprod. 31, 291-307 (2018).

[23] Miller, C. et al. Variation in expression of the HECT e3 Ligase UPL3 modulates LEC2 levels, seed size, and crop yields in Brassica napus. Plant Cell 31, 2370-2385 (2019).

[24] Mu, J. et al. LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in arabidopsis. Plant Physiol. 148, 1042-1054 (2008).

[25] Zhang, F. et al. Transcriptomic and proteomic analysis reveals mechanisms of embryo abortion during chrysanthemum cross breeding. Sci. Rep. 4, 6536 (2015).

[26] Zhang, F. et al. Identification of microRNAs and their targets associated with embryo abortion during chrysanthemum cross breeding via high-throughput sequencing. PLoS ONE 10, e124371 (2015).

[27] Zhang, F. et al. Chromosome doubling to overcome the chrysanthemum cross barrier based on insight from transcriptomic and proteomic analyses. BMC Genomics 17, 585 (2016).

[28] Sakamoto, H. et al. Arabidopsis Cys2/His2-Type Zinc-Finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol. 136, 2734-2746 (2004).

[29] Pelletier, J. M. et al. LEC1 sequentially regulates the transcription of genes involved in diverse developmental processes during seed development. Proc. Natl Acad. Sci. USA 114, E6710-E6719 (2017).

[30] Lotan, T. et al. Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell 93, 1195-1205 (1998).

[31] Gnesutta, N. et al. Crystal structure of the arabidopsis thaliana L1L/NF-YC3 histone-fold dimer reveals specificities of the LEC1 family of NF-Y subunits in plants. Mol. Plant 10, 645-648 (2017).

[32] Jo, L., Pelletier, J. M. & Harada, J. J. Central role of the LEAFY COTYLEDON1 transcription factor in seed development. J. Integr. Plant Biol. 61, 564-580 (2019).

[33] Bryant, F. M. et al. Basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 transactivates DELAY of GERMINATION1 to establish primary seed dormancy in arabidopsis. Plant Cell 31, 1276-1288 (2019).

[34] Hundertmark, M. & Hincha, D. K. LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics 9, 118 (2008).

[35] Magwanga, R. O. et al. Characterization of the late embryogenesis abundant (LEA) proteins family and their role in drought stress tolerance in upland cotton. BMC Genet. 19, 6 (2018).

[36] Delahaie, J. et al. LEA polypeptide profiling of recalcitrant and orthodox legume seeds reveals ABI3-regulated LEA protein abundance linked to desiccation tolerance. J. Exp. Bot. 64, 4559-4573 (2013).

[37] Galau, G. A., Jakobsen, K. S. & Hughes, D. W. The controls of late dicot embryogenesis and early germination. Physiol. Plant. 81, 280-288 (1991).

[38] Vicient, C. M. et al. Changes in gene expression in the leafy cotyledon1 (lec1) and fusca3 (fus3) mutants of Arabidopsis thaliana L. J. Exp. Bot. 347, 995-1003 (2000).

[39] Bies-Etheve, N. et al. Importance of the B2 domain of the Arabidopsis ABI3 protein for Em and 2S albumin gene regulation. Plant Mol. Biol. 40, 1045-1054 (1999).

[40] Delseny, M. et al. Late Embryogenesis Abundant (LEA) protein gene regulation during Arabidopsis seed maturation. J. Plant Physiol. 158, 419-427 (2001).

[41] Chileh, T. et al. Characterization of the 11S globulin gene family in the castor plant ricinus communis l. J. Agr. Food Chem. 58, 272-281 (2010).

[42] Nguyen, T. et al. A role for seed storage proteins in Arabidopsis seed longevity. J. Exp. Bot. 66, 6399-6413 (2015).

[43] Lee, H. et al. Arabidopsis LEAFY COTYLEDON1 represents a functionally specialized subunit of the CCAAT binding transcription factor. Proc. Natl Acad. Sci. USA 100, 2152-2156 (2003).

[44] Petroni, K. et al. The promiscuous life of plant NUCLEAR FACTOR Y transcription factors. Plant Cell 24, 4777-4792 (2013).

[45] Simkin, A. J. et al. Oleosin gene family of Coffea canephora: quantitative expression analysis of five oleosin genes in developing and germinating coffee grain. J. Plant Physiol. 163, 691-708 (2006).

[46] Guo, Y. H. et al. GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5. N. Phytol. 183, 62-75 (2009).

[47] Long, Y. et al. Arabidopsis BIRD zinc finger proteins jointly stabilize tissue boundaries by confining the cell fate regulator SHORT-ROOT and contributing to fate specification. Plant Cell 27, 1185-1199 (2015).

[48] Wang, D. et al. Genome-wide analysis of CCCH zinc finger family in Arabidopsis and rice. BMC Genomics 9, 44 (2008).

[49] Li, Z. & Thomas, T. L. PEI1, an embryo-specific zinc finger protein gene required for heart-stage embryo formation in arabidopsis. Plant Cell 10, 383-398 (1998).

[50] Ming et al. Over-expression of a C3H-type zinc finger gene contributes to salt stress tolerance in transgenic broccoli plants. Plant Cell Tissue Organ Cult. 130, 239-254 (2017).

[51] Wang, L. et al. OsLIC, a novel CCCH-Type zinc finger protein with transcription activation, mediates rice architecture via brassinosteroids signaling. PLoS ONE 3, e3521 (2008).

[52] Franke, R. et al. Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis. Plant J. 30, 47-59 (2010).

[53] Jiang, A. et al. Genome-Wide analysis of the C3H zinc finger transcription factor family and drought responses of members in aegilops tauschii. Plant Mol. Biol. Rep. 32, 1241-1256 (2014).

[54] Jiao, Z. et al. Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula. BMC Plant Biol. 20, 401 (2020).

[55] Hall, T. M. Multiple modes of RNA recognition by zinc finger proteins. Curr. Opin. Struc. Biol. 15, 367-373 (2005).

[56] Zhang, C. et al. Genome-wide analysis of the CCCH zinc finger gene family in medicago truncatula. Plant Cell Rep. 32, 1543-1555 (2013).

[57] Wang, G. et al. An expression analysis of 57 transcription factors derived from ESTs of developing seeds in Maize (Zea mays). Plant Cell Rep. 29, 545-559 (2010).

[58] Dure, L. et al. Common amino acid sequence domains among the LEA proteins of higher plants. Plant Mol. Biol. 12, 475-486 (1989).

[59] Xu, M. et al. Transcriptomic analysis of the grapevine LEA gene family in response to osmotic and cold stress reveals a key role for VamDHN3. Plant Cell Physiol. 61, 775-786 (2020).

[60] Boulard, C. et al. LEC1 (NF-YB9) directly interacts with LEC2 to control gene expression in seed. Biochim. Biophys. Acta 1861, 443-450 (2018).

[61] Liu, S. et al. In vitro induced tetraploid of Dendranthema nankingense (Nakai) Tzvel. shows an improved level of abiotic stress tolerance. Sci. Hortic. 127, 411-419 (2011).

[62] Sun, C. et al. Interspecific hybrids between Chrysanthemum grandiflorum (Ramat.) Kitamura and C. Indicum (L.) Des Moul. and their drought tolerance evaluation. Euphytica 174, 51-60 (2010).

[63] Guan, Y. et al. Overexpression of the CmJAZ1-like gene delays flowering in Chrysanthemum morifolium. Hortic. Res. 8, 87 (2021).

[64] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR. Methods 25, 402-408 (2002).

[65] Kumar, S., Stecher, G. & Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870-1874 (2016).

[66] Zhou, F. et al. Correction: Corrigendum: D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling. Nature 532, 406-410 (2013).

[67] Curtis, M. D. & Grossniklaus, U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462-469 (2003).

[68] Schwab, R. et al. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18, 1121-1133 (2006).

[69] Teng, N. et al. A high-efficiency Agrobacterium-mediated transformation method via vacuum infiltration of chrysanthemum stem internodes. China. Patent 2017113822474.

[70] Wang, C. Q. et al. BBX19 interacts with CONSTANS to repress FLOWERING LOCUS T transcription, defining a flowering time checkpoint in arabidopsis. Plant Cell 26, 3589-3602 (2014).

[71] Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011).

[72] Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359 (2012).

[73] Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

[74] Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).